The American College of Obstetrics and Gynecology (ACOG) Practice Bulletin Number 77 published in 2007 supports that first trimester aneuploidy risk assessment, based on nuchal translucency measurement and surrogate biochemical markers to screen for Down syndrome, for all pregnant women (ACOG Practice Bulletin No. 77, Obstet Gynecol 109:217-227 [2007]). These screening tests can only provide a risk determination that is inconclusive and has non-optimal determination and high false positive rates. Today, only invasive methods including chorionic villus sampling (CVS), amniocentesis or cordocentesis provide definite genetic information about the fetus, but these procedures are associated with risks to both mother and fetus (Odibo et al., Obstet Gynecol 112:813-819 [2008]; Odibo et al., Obstet Gynecol 111:589-595 [2008]; Evans and Wapner, Semin Perinatol 29:215-218 [2005]). Therefore, a non-invasive means to obtain definite information on fetal chromosomal status is desirable.
Massively parallel DNA sequencing of cfDNA obtained from the maternal plasma yields millions of short sequence tags that can be aligned and uniquely mapped to sites from a reference human genome, and the counting of the mapped tags can be used to determine the over- or under-representation of a chromosome (Fan et al., Proc Natl Acad Sci USA 105:16266-16271 [2008]; Voelkerding and Lyon, Clin Chem 56:336-338 [2010]). However, the depth of sequencing and subsequent counting statistics determines the sensitivity of determination for fetal aneuploidy. The requirement for an optimized algorithm to determine chromosomal aneuploidies in maternal plasma samples is underscored by the apparent inability to determine more than one type of trisomy in a population of test samples (Chiu et al., BMJ 342, c7401 [2011]; Ehrich et al., Am J Obstet Gynecol 2014:205 e1 [2011]).
The limitations of the existing methods underlie the need for optimal noninvasive methods that would provide any or all of the specificity, sensitivity, and applicability to reliably diagnose chromosomal aneuploidies for prenatal diagnoses and for the diagnoses and monitoring of medical conditions associated with copy number changes.
The present invention fulfills some of the above needs and in particular offers an advantage in providing a reliable method having sufficient sensitivity to determine single or multiple chromosomal aneuploidies, and which verifies that the correct determination is made.